Guidelines For The Laboratory Use of Autoantibody Tests
Guidelines For The Laboratory Use of Autoantibody Tests
Guidelines For The Laboratory Use of Autoantibody Tests
Key Words: Antinuclear antibodies; Autoimmune rheumatic disease; Guidelines; Dermatopolymyositis; Systemic sclerosis; Systemic lupus
because weak ANA reactivity is present in many nonrheumatic Among the nuclear patterns, the most frequent consist
patients and even in “healthy” control subjects. of homogeneous/peripheral fluorescence (DNA, deoxyri-
Systemic autoimmune diseases are characterized by bonucleoprotein, histones) and speckled fluorescence (RNP,
the presence of high titers of serum autoantibodies to Sm, Ro/SSA, La/SSB); the relatively frequent patterns
intracellular proteins and nucleic acids.21 These autoanti- include the centromeric (CENP-A, CENP-B, CENP-C),
bodies are generally referred to as antinuclear antibodies nucleolar (PM/Scl, nucleolin, fibrillarin, RNA polymerase I,
owing to their predominant reactivity with nuclear anti- human upstream binding factor), and the diffuse grainy
gens, but they also include anticytoplasmic antibodies. (topoisomerase I or Scl70) patterns, while the rarer pictures
The test for ANAs has a very high diagnostic sensitivity, include the proliferating cell nuclear antigen (PCNA) or
such that it is considered the best test for the screening for cyclin, nuclear dots (coilin, Sp-100), and nuclear membrane
these diseases.7,22 (lamins A, B, and C; glycoprotein 210) patterns.
As most ANAs have an IgG isotype, and the detection Cytoplasmic patterns are relatively less frequent and
and enables the establishment of different cutoff levels, each would classify virtually all patients with SLE, systemic scle-
one with its own characteristics of sensitivity and specificity; rosis, and Sjögren syndrome as positive for ANA.20
however, this method also is bound to a subjective definition High autoantibody titers (>1:160) are present in sick
of the end point and, thus, is poorly reproducible, as subjects, but in only 5% of healthy people; this threshold is a
evidenced by the wide scattering of the titers obtained in little less sensitive but more specific.20 Indeed, at an end point
numerous collaborative studies.20,31,32 titer less than 1:160, the follow-up secondary tests will yield
The analytic imprecision or inaccuracy related to the use positive results in fewer than 5% of the cases.36,40 Intermediate
of dilutions makes it preferable to use calibrators adjusted titers may be present in about 20% of the healthy population
against an international reference standard (World Health and in a small percentage of sick subjects. For these reasons:
Organization [WHO] International Reference Preparation Titers of 1:40 and 1:160 (or concentrations of 5 and 20
[IRP] 66/233, homogeneous pattern) and to express the IU, respectively) are considered decision-making levels that
results in international units of concentration (IU/mL).23,33 require different operative algorithms:
for both screening and detection of total ANAs. While such became evident that the DNA molecule presented several
EIA-ANA tests are less labor intensive and can be used to epitopes and, therefore, that the anti-DNA antibodies
screen and detect a panel of common specific autoantibodies included a heterogeneous group of immunoglobulins with
usually observed in systemic rheumatic diseases, they are not different specificities. The most commonly identified anti-
100% sensitive when compared with IIF-ANA43-47; in addi- bodies are those directed against single-stranded DNA
tion, the EIA-ANA test may fail to detect certain ANAs with (ssDNA), whose antigenic determinants seem to be localized
atypical or rare immunofluorescent patterns.39 in the basic purine and pyrimidine sequences; the antibodies
Moreover, there are remarkable qualitative differences directed against dsDNA instead recognize epitopes localized
between the different reagents48; some kits contain only a along the deoxyribose-phosphate backbone.49
narrow assortment of antigens, whereas other include whole Compared with the lack of specificity of anti-ssDNA
cell extracts and others whole cell extracts spiked with scarcely antibodies, anti-dsDNA antibodies are highly specific for
represented antigens. Furthermore, some manufacturers use SLE and are present in affected subjects with a prevalence
however, it may also detect antibodies with low avidity of classification has been demonstrated. At present, the
having uncertain clinical significance.55 Therefore, positive autoantibodies with these characteristics are anti-Ro/SSA,
results require subsequent confirmation by IIF, which has a anti-La/SSB, anti-Sm, anti-U1RNP, anti-Scl70, anti–Jo-1,
higher specificity. anti–CENP-B, anti-rRNP, and antinucleosome (chromatin);
Quantification of anti-dsDNA antibodies is useful for therefore:
the clinical management of the patient with SLE. Several In the diagnostic phase, extend the detection of antinu-
studies have documented a close relationship between anti- clear specific autoantibodies to the following autoantigens:
body titer and disease activity, particularly lupus nephritis56; Ro/SSA, La/SSB, Sm, U1RNP, Scl70 (topoisomerase I), Jo-1
an increase in the antibody concentration may precede flares (histidyl-tRNA synthetase), CENP-B, rRNP, and nucleosome
of disease by a few weeks.57,58 In general, for patients with (chromatin).
relatively active disease, anti-dsDNA can be tested every 6 to Serum antinuclear specific antibodies can be detected
12 weeks, whereas for those with less active disease, a with several techniques currently used in the laboratory:
strategy for the search for antinuclear specific antibodies must References
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